1. Field of the Invention
The present invention relates to antibody binding assays and radiolabeling kits, lyophilized cell preparations, reagents and protocols for testing the clinical efficacy of therapeutic antibodies for the treatment/imaging of tumors and tumor cells. Specifically, the kits of the present invention are used for making and evaluating radiolabeled antibody conjugates that will be used for the treatment and imaging of B-cell lymphoma tumors by targeting the B cell surface antigen BP35 (“CD20”).
2. Technology Background
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The immune system of vertebrates (for example, primates, which include humans, apes, monkeys, etc.) consists of a number of organs and cell types which have evolved to: accurately and specifically recognize foreign microorganisms (“antigen”) which invade the vertebrate-host; specifically bind to such foreign microorganisms; and, eliminate/destroy such foreign microorganisms. Lymphocytes, as well as other types of cells, are critical to the immune system. Lymphocytes are produced in the thymus, spleen and bone marrow (adult) and represent about 30% of the total white blood cells present in the circulatory system of humans (adult).
There are two major sub-populations of lymphocytes: T cells and B cells. T cells are responsible for cell mediated immunity, while B cells are responsible for antibody production (humoral immunity). However, T cells and B cells can be considered as interdependent—in a typical immune response, T cells are activated when the T cell receptor binds to fragments of an antigen that are bound to major histocompatability complex (“MHC”) glycoproteins on the surface of an antigen presenting cell; such activation causes release of biological mediators (“interleukins”) which, in essence, stimulate B cells to differentiate and produce antibody (immunoglobulins”) against the antigen.
Each B cell within the host expresses a different antibody on its surface—thus one B cell will express antibody specific for one antigen, while another B cell will express antibody specific for a different antigen. Accordingly, B cells are quite diverse, and this diversity is critical to the immune system. In humans, each B cell can produce an enormous number of antibody molecules (i.e. about 107 to 108). Such antibody production most typically ceases (or substantially decreases) when the foreign antigen has been neutralized. Occasionally, however, proliferation of a particular B cell will continue unabated; such proliferation can result in a cancer referred to as “B cell lymphoma.”
T cells and B cells both comprise cell surface proteins which can be utilized as “markers” for differentiation and identification. One such human B cell marker is the human B lymphocyte-restricted differentiation antigen Bp35, referred to as “CD20.” CD20 is expressed during early pre-B cell development and remains until plasma cell differentiation. Specifically, the CD20 molecule may regulate a step in the activation process which is required for cell cycle initiation and differentiation and is usually expressed at very high levels on neoplastic (“tumor”) B cells. CD20, by definition, is present on both “normal” B cells as well as “malignant” B cells, i.e., those B cells whose unabated proliferation can lead to B cell lymphoma. Thus, the CD20 surface antigen has the potential of serving as a candidate for “targeting” of B cell lymphomas.
In essence, such targeting can be generalized as follows: antibodies specific to the CD20 surface antigen of B cells are, e.g., injected into a patient. These anti-CD20 antibodies specifically bind to the CD20 cell surface antigen of (ostensibly) both normal and malignant B cells; the anti-CD20 antibody bound to the CD20 surface antigen may lead to the destruction and depletion of neoplastic B cells. Additionally, chemical agents or radioactive labels having the potential to destroy the tumor can be conjugated to the anti-CD20 antibody such that the agent is specifically “delivered” to, e.g., the neoplastic B cells. Irrespective of the approach, a primary goal is to destroy the tumor: the specific approach can be determined by the particular anti-CD20 antibody which is utilized and, thus, the available approaches to targeting the CD20 antigen can vary considerably.
For example, attempts at such targeting of CD20 surface antigen have been reported. Murine (mouse) monoclonal antibody 1F5 (an anti-CD20 antibody) was reportedly administered by continuous intravenous infusion to B cell lymphoma patients. Extremely high levels (>2 grams) of 1F5 were reportedly required to deplete circulating tumor cells, and the results were described as being “transient.” Press et al., “Monoclonal Antibody 1F5 (Anti-CD20) Serotherapy of Human B-Cell Lymphomas,” Blood 69/2:584-591 (1987).
A potential problem with this approach is that non-human monoclonal antibodies (e.g., murine monoclonal antibodies) typically lack human effector functionality, i.e., they are unable to, inter alia, mediate complement dependent lysis or lyse human target cells through antibody dependent cellular toxicity or Fc-receptor mediated phagocytosis. Furthermore, non-human monoclonal antibodies can be recognized by the human host as a foreign protein; therefore, repeated injections of such foreign antibodies can lead to the induction of immune responses leading to harmful hypersensitivity reactions. For murine-based monoclonal antibodies, this is often referred to as a Human Anti-Mouse Antibody response, or “HAMA” response. Additionally, these “foreign” antibodies can be attacked by the immune system of the host such that they are, in effect, neutralized before they reach their target site.
Lymphocytes and lymphoma cells are inherently sensitive to radiotherapy. Therefore, B cell malignancies are attractive targets for radioimmunotherapy (RIT) for several reasons: the local emission of ionizing radiation of radiolabeled antibodies may kill cells with or without the target antigen (e.g., CD20) in close proximity to antibody bound to the antigen; penetrating radiation, i.e., beta emitters, may obviate the problem of limited access to the antibody in bulky or poly vascularized tumors; and, the total amount of antibody required may be reduced. The radionuclide emits radioactive particles which can damage cellular DNA to the point where the cellular repair mechanisms are unable to allow the cell to continue living; therefore, if the target cells are tumors, the radioactive label beneficially kills the tumor cells. Radiolabeled antibodies, by definition, include the use of a radioactive substance which may require the need for precautions for both the patient (i.e., possible bone marrow transplantation) as well as the health care provider (i.e., the need to exercise a high degree of caution when working with radioactivity).
Therefore, an approach at improving the ability of murine monoclonal antibodies to effect the treatment of B-cell disorders has been to conjugate a radioactive label to the antibody such that the label or toxin is localized at the tumor site. Toxins (i.e., chemotherapeutic agents such as doxorubicin or mitomycin C) have also been conjugated to antibodies. See, for example, PCT published application WO 92/07466 (published May 14, 1992).
“Chimeric” antibodies, i.e., antibodies which comprise portions from two or more different species (e.g., mouse and human) have been developed as an alternative to “conjugated” antibodies. Mouse/human chimeric antibodies have been created, and shown to exhibit the binding characteristics of the parental mouse antibody, and effector functions associated with the human constant region. See e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Shoemaker et al., U.S. Pat. No. 4,978,745; Beavers et al., U.S. Pat. No. 4,975,369; and Boss et al., U.S. Pat. No. 4,816,397 all of which are incorporated by reference herein. Generally these chimeric antibodies are constructed by preparing a genomic gene library from DNA extracted from pre-existing murine hybridomas. Nishimura et al. (1987) Cancer Research 47: 999. The library is then screened for variable region genes from both heavy and light chains exhibiting the correct antibody fragment rearrangement patterns. The cloned variable region genes are then ligated into an expression vector containing cloned cassettes of the appropriate heavy or light chain human constant region gene. The chimeric genes are then-expressed in a cell line of choice, usually a murine myeloma line.
For example, Liu, A. Y., et al., “Production of a Mouse-Human Chimeric Monoclonal Antibody to CD20 with Potent Fc-Dependent Biologic Activity”, J. Immun. 139/10:3521-3526 (1987), describes a mouse/human chimeric antibody directed against the CD20 antigen. See also, PCT Publication No. WO 88/04936. However, no information is provided as to the ability, efficacy or practicality of using Liu's chimeric antibodies for the treatment of B cell disorders in the reference.
It is noted that in vitro functional assays (e.g. complement dependent lysis (“CDC”); antibody dependent cellular cytotoxicity (“ADCC”), etc.) cannot inherently predict the in vivo capability of any antibody to destroy or deplete target cells expressing the specific antigen. See, for example, Robinson, R. D., et al., “Chimeric mouse-human anti-carcinoma antibodies that mediate different antitumor cell biological activities,” Hum. Antibod. Hybridomas, 2:84-93 (1991) (chimeric mouse-human antibody having undetectable ADCC activity). Therefore, the potential therapeutic efficacy of antibodies can only truly be assessed by in vivo experimentation.
To this end application Ser. No. 08/475,813 (issued as U.S. Pat. No. 6,682,734 B1 on Jan. 27, 2004), Ser. No. 08/475,815 (issued as U.S. Pat. No. 6,399,061 B1 on Jun. 4, 2002), and Ser. No. 08/478,967 (issued as U.S. Pat. No. 5,843,439 on Dec. 1, 1998), herein incorporated by reference in their entirety, disclose radiolabeled ant-CD20 conjugates for diagnostic “imaging” of B cell lymphoma tumors before administration of therapeutic antibody. “In2B8” conjugate comprises a murine monoclonal antibody, 2B8, specific to human CD20 antigen, that is attached to Indium [111] (111In) via a bifuncional chelator, i.e., MX-DTPA (diethylenetriaminepentaacetic acid), which comprises a 1:1 mixture of 1-isothiocyanatobenzyl-3-methyl DTPA and 1methyl-3-isothiocyanatobenzyl-DTPA. Indium-[111] is selected as a diagnostic radionuclide because it emits gamma radiation and finds prior usage as an imaging agent.
Patents relating to chelators and chelator conjugates are known in the art. For instance, U.S. Pat. No. 4,831,175 of Gansow is directed to polysubstituted diethylenetriaminepentaacetic acid chelates and protein conjugates containing the same, and methods for their preparation. U.S. Pat. Nos. 5,099,069, 5,246,692, 5,286,850, and 5,124,471 of Gansow also relate to polysubstituted DTPA chelates. These patents are incorporated herein in their entirety.
The specific bifunctional chelator used to facilitate chelation in application Ser. No. 08/475,813, Ser. No. 08/475,815 and Ser. No. 08/478,967 was selected as it possesses high affinity for trivalent metals, and provides for increased tumor-to-non-tumor ratios, decreased bone uptake, and greater in vivo retention of radionuclide at target sites, i.e., B-cell lymphoma tumor sites. However, other bifunctional chelators are known in the art and may also be beneficial in tumor therapy.
Also disclosed in application Ser. Nos. 08/475,813 (issued as U.S. Pat. No. 6,682,734, B1 on Jan. 27, 2004), Ser No. 08/475,815 (issued as U.S. Pat. No. 6,399,061 B1 on Jun. 4, 2002), and antibodies for the targeting and destruction of B cell lymphomas and tumor cells. In particular, the Y2B8 conjugate comprises the same anti-human CD20 murine monoclonal antibody, 2B8, attached to yttrium-[90] (90Y) via the same bifunctional chelator. This radionuclide was selected for therapy for several reasons. The 64 hour half-life of 90Y is long enough to allow antibody accumulation by the tumor and, unlike e.g. 131I, it is a pure beta emitter of high energy with no accompanying gamma irradiation in its decay, with a range of 100 to 1000 cell diameters. The minimal amount of penetrating radiation allows for outpatient administration of 90Y-labeled antibodies. Furthermore, internalization of labeled antibodies is not required for cell killing, and the local emission of ionizing radiation should be lethal for adjacent tumor cells lacking the target antigen.
Because the 90Y radionuclide was attached to the 2B8 antibody using the same bifunctional chelator molecule MX-DTPA, the Y2B8 conjugate possesses the same advantages discussed above, e.g., increased retention of radionuclide at a target site (tumor). However, unlike 111In, it cannot be used for imaging purposes due to the lack of gamma radiation associated therewith. Thus, a diagnostic “imaging” radionuclide, such as 111In, can be used for determining the location and relative size of a tumor prior to and/or following administration of therapeutic chimeric or 90Y-labeled antibodies for the purpose of tumor reduction. Additionally, indium-labeled antibody enables dosimetric assessment to be made.
Depending on the intended use of the antibody, i.e., as a diagnostic or therapeutic reagent, other radiolabels are known in the art and have been used for similar purposes. For instance, radionuclides which have been used in clinical diagnosis include 131I, 125I, 123I, 99Tc, 67Ga as well as 111In. Antibodies have also been labeled with a variety of radionuclides for potential use in targeted immunotherapy (Peirersz et al. (1987) The use of monoclonal antibody conjugates for the diagnosis and treatment of cancer. Immunol. Cell Biol. 65: 111-125). These radionuclides include 188Re and 186Re as well as 90Y, and to a lesser extent 199Au and 67Cu. I-[131] has also been used for therapeutic purposes. U.S. Pat. No. 5,460,785 provides a listing of such radioisotopes and is herein incorparted by reference.
As reported in copending application Ser. Nos. 08/475,813 (issued as U.S. Pat. No. 6,682,734 B1 on Jan. 27, 2004), Ser .No. 08/475,815 (issued as U.S. Pat. No. 6,399,061 B1 on Jun. 4, 2002), and Ser. No. 08/478,967, (issued as U.S. Pat. No. 5,843,439, on Dec. 1, 1998),administration of the radiolabeled Y2B8 conjugate, as well as unlabeled chimeric anti-CD20 antibody, resulted in significant tumor reduction in mice harboring a B cell lymphoblastic tumor. Moreover, human clinical trials reported therein showed significant B cell depletion in lymphoma patients infused with chimeric anti-CD20 antibody. In fact, chimeric 2B8 has recently been heralded the nation's first FDA-approved anti-cancer monoclonal antibody underthe name of Rituxan®. Thus, at least one chimeric anti-CD20 antibody has been shown to demonstrate therapeutic efficacy in the treatment of B cell lymphoma.
In addition, U.S. application Ser. No. 08/475,813 (issued as U.S. Pat. No. 6,682,734, B1 on Jan. 27, 2004), herein incorporated by reference, discloses sequential administration of Rituxan®, a chimeric anti-CD20, with both or either indium-labeled or yttrium-labeled murine monoclonal antibody. Although the radiolabeled antibodies used in these combined therapies are murine antibodies, initial treatment with chimeric anti-CD20 sufficiently depletes the B cell population such that the HAMA response is decreased, thereby facilitating a combined therapeutic and diagnostic regimen.
Thus, in this context of combined immunotherapy, murine antibodies may find particular utility as diagnostic reagents. Moreover, it was shown in U.S. application Ser. No. 08/475,813 (issued as U.S. Pat. No. 6,682,734 B1 on Jan. 27, 2004) that a therapeutically effective dosage of the yttrium-labeled anti-CD20 antibody following administration of Rituxan® is sufficient to    (a) clear any remaining peripheral blood B cells not cleared by the chimeric anti-CD20 antibody;    (b) begin B cell depletion from lymph nodes; or (c) begin B cell depletion from other tissues.
Thus, conjugation of radiolabels to cancer therapeutic antibodies provides a valuable clinical tool which may be used to assess the potential therapeutic efficacy of such antibodies, create diagnostic reagents to monitor the progress of treatment, and devise additional therapeutic reagents which may be used to enhance the initial tumor-killing potential of the chimeric antibody. Given the proven efficacy of an anti-CD20 antibody in the treatment of non-Hodgkin's lymphoma, and the known sensitivity of lymphocytes to radioactivity, it would be highly advantageous for such therapeutic antibodies to become commercially available in kit form whereby they may be readily modified with a radiolabel and administered directly to the patient in the clinical setting.
Although there exist many methods and reagents for accomplishing radiolabeling of antibodies, what is lacking in the art is a convenient vehicle for placing these reagents in the clinical setting, in a way that they may be easily produced and administered to the patient before significant decay of the radiolabel or significant destruction of the antibody due to the radiolabel occurs. The lack of such convenient means to commercialize this valuable technology could be due to the poor incorporation efficiencies demonstrated by some known labeling protocols, and the subsequent need to column purify the reagent following the radiolabeling procedure. The delay in development of such kits might also in part be due to the previously lack of accessibility to pure commercial radioisotopes which may be used to generate efficiently labeled products absent subsequent purification. Alternatively, perhaps the reason such kits are generally unavailable is the actual lack of antibodies which have been able to achieve either the approval or the efficacy that Rituxan® has achieved for the treatment of lymphoma in human patients.
For instance, as discussed in U.S. Pat. No. 4,636,380, herein incorporated by reference, it has been generally believed in the scientific community that for a radiopharmaceutical to find clinical utility, it must endure a long and tedious separation and purification process. Indeed, injecting unbound radiolabel into the patient would not be desirable. The need for additional purification steps renders the process of radiolabeling antibodies in the clinical setting an impossibility, particularly for doctors who have neither the equipment nor the time to purify their own therapeutics.
Furthermore, radiolabeled proteins may be inherently unstable, particularly those labeled with radiolytic isotopes such as 90Y, which have the tendency to cause damage to the antibody the longer they are attached to it in close proximity. In turn, such radiolysis causes unreliable efficiency of the therapeutic due to loss of radiolabel and/or reduced binding to the target antigen, and may lead to undesired immune responses directed at denatured protein. Yet without the facilities for labeling and purifying the antibodies on site, clinicians have had no choice but to order therapeutic antibodies already labeled, or have them labeled off site at a related facility and transported in following labeling for administration to the patient. All such manipulations add precious time to the period between labeling and administration, thereby contributing to the instability of the therapeutic, while in effect decreasing the utility of radiolabeling kits in the clinical setting.
Others have tried unsuccessfully to develop antibody radiolabeling kits that would be proficient enough to forego a separate purification step of the antibody. For instance, Cytogen has recently launched a commercial kit for radiolabeling a murine monoclonal antibody directed to tumor-associated glycoprotein TAG-72. However, Cytogen's antibody is particularly unamenable to a kit formulation due to the tendency to develop particulates during storage which must later be removed by a further filtration step. Moreover, Cytogen's antibody has caused adverse reactions in patients due to a HAMA responses.
Others have claimed to have developed radiolabeling protocols which would be amenable to kit format in that a separate purification step would not be required (Richardson et al. (1987) Optimization and batch production of DTPA-labeled antibody kits for routine use in 111In immunoscintography. Nuc. Med. Commun. 8: 347-356; Chinol and Hnatowich (1987) Generator-produced yttrium-[90] for radioimmunotherapy. J. Nucl. Med. 28(9): 1465-1470). However, such protocols were not able to achieve the level of incorporation that the present inventors have achieved using the protocols disclosed herein, which have resulted in incorporation efficiencies of at least 95%. Such a level of incorporation provides the added benefit of increased safety, in that virtually no unbound label will be injected into the patient as a result of low radioincorporation.
The protocols included in the kits of the present invention allow rapid labeling which may be affected in approximately a half an hour or as little as five minutes depending on the label. Moreover, the kit protocols of the present invention have a labeling efficiency of over 95% thereby foregoing the need for further purification. By foregoing the need for further purification, the half-life of the radiolabel and the integrity of the antibody is reserved for the therapeutic purpose for which it is labeled.
The present application discloses convenient kits and methods whereby diagnostic and therapeutic antibodies may be radiolabeled and administered to a patient in a reproducible, reliable and convenient manner. The kits of the present invention transform the process of radiolabeling antibodies into a hassle-free, worry-free standardized process, which greatly facilitates patient treatment protocols. The present kits provide advantages over the prior art in that the optimum parameters for labeling and administering therapeutic or diagnostic have been determined, thereby-reducing the cost of goods. Since the kits described herein provide the optimum parameters according to the particular label, use of a kit designed for a particular label will also minimize cannibalization, i.e., which occurs when an inappropriate kit is used for a particular label. Avoiding cannibalization in turn also provides for optimum labeling efficiency. Moreover, the protocols and sterile; pyrogen-free ingredients included with each kit make for a more user-friendly process, since sterility, pyrogen testing and post-labeling purification of the reagents are obviated.